Over the past two decades, there has been a rapid emergence of plant genetic engineering technology coupled with major improvements in the development of large-scale plant cell culture processes for the production of useful secondary metabolites. From 1995 (Moffat, 1995; Ma et al., 2003), such plant cell suspension cultures are increasingly used as a valuable host cell system for the expression of recombinant proteins.
Auxin-induced callus tissue or suspensions, in spite of their single tissue origin, usually contains cells with a variety of phenotypes. Thus, transgenic lines developed from such cell types are usually highly heterogeneous with inconsistent expression levels. Therefore, clones producing many useful secondary metabolites have been obtained from single protoplasts, i.e., a high shikonin-producing cell clone prepared from Lithospermum erythrorhizon protoplasts (Maeda et al., 1983).
So far, it has been necessary to form protoplasts to disaggregate cells not only for cell selection but also for electro-poration/PEG mediated transformation of cultured plant cells. Protoplast preparation has been required for the isolation of single cell clones from plant tissues. However, it is usually difficult for protoplasts to regenerate their normal walls, because isolated protoplasts are usually arrested and reluctant to divide (Hahne and Hoffmann, 1984). In many studies on cultured protoplasts, the first and major polysaccharide generated is callose, which is composed of 1,3-, 8-glucopyranoses (Klein et al., 1981).
Wounded or stressed plants often secrete masses of this glucan into periplasmic spaces (Currier, 1957). During early stages of wall regeneration, binding between cellulose and xyloglucan is not as strong as it is in intact plants (Hayashi et al., 1986). Since the macromolecular organization of xyloglucan and cellulose in the primary cell walls appears to be responsible for the strength and extensibility (Hayashi and Maclachlan, 1984), the deposition of xyloglucan as well as cellulose around protoplasts appears to be critical to their division and growth potential. This prerequisite stalls the division of protoplast, thus increasing the time to regenerate into normal cells with the cellular characteristics of the parental lines.
An ongoing technical challenge, therefore, in the field of plant cell culture is to isolate single viable cells that can be cloned from plant tissue in culture (Bourgin, 1983; Tabata et al., 1976). In suspension culture, non-uniform cell aggregates always form, and each such aggregates contain up to hundred cells. Nothing is known about the linkage between these aggregated cells, and there has been no report identifying a single enzyme that can dissociate cell aggregates and maintain them as single cells in vitro with intact cell walls.
Pectin's role in cell adhesive properties was suggested in several reports, but such a link was established relatively more recently (Bouton et al., 2002). In addition, strong reductions in cell adhesive properties were reported (Sterling et al., 2006).
The qual-1 mutants showed detached single root cells (Bouton et al., 2002). The reduced pectin content was corroborated further by immunofluorescence experiments using antibodies raised against specific pectic epitopes. These observations suggest that the encoded enzyme may be involved in the synthesis of pectic polysaccharides and clearly indicated pectin is involved in the adhesive properties of plant cells.
Thus, disrupting pectin synthesis to remove cell adhesive properties can facilitate cell separation. Single cell isolation with one time pectin degrading enzyme treatments (Naill, 2005) has been reported to aid isolation of single cells in Taxus cell suspension cultures. Such Taxus single cells are used to screen for elite clonal lines with higher level of Taxol production. However, this method is not useful in the maintenance of single suspension cells in the continuous presence of the enzyme in the medium. Also, the highest single cell yield with such short pulse treatment of enzymes or combination of enzymes was only 17.1% to 34.4% (Naill, 2005). Continuous pectinase treatment in rice suspensions have only resulted in fine suspension aggregates at 0.005% concentrations but has not helped to maintain the suspension as single cells (Lee et al., 2004). Prolonged treatments of combination of enzymes, pectinase and cellulase for more than 8 hours have resulted in cell lysis (Naill, 2005).
Enhancement of cell separation in suspension cultures of soybean cells has been reported to be enhanced in the presence of colchicine (Umetsu et al., 1975). For cell separation the alkaloid was added to culture medium at lower concentrations (0.1-1.0 mM) than those (5-20 mM) for the production of chromosomal polyploidy. Nonetheless, colchicine inhibits mitosis in plant and animal cells (Lewin, 1980). Colchicine binds to tubulin and prevents the assembly of microtubules. Therefore, to obtain cell separation, the colchicines concentration and treatment time should be as low as possible.
Colchicine alkaloids have been used for synchronization of growth in cultured animal cells where the alkaloids are usually added at 0.5 mM, and the cells should be arrested within a few hours, before the mitosis. Although the morphogenic effect is quite similar to that in animal cells, plant cells can divide during growth in the presence of colchicine at 0.1 mM (Umetsu et al., 1975). Cell viability decreased after 4 days of culture of soybean suspension cells in 1 mM colchicine. In addition, only 44.8% of the cells were viable in these treatments, but it was possible to keep them dividing unlike in animal cells.
The use of tubulin depolimerization inhibitors or on the oligosaccharins in the maintenance of single cell suspension in plant in vitro cultures has been investigated. The literature has some information as early as 1975 regarding using colchicines for cell separation; see References section, below. Tubulin inhibitors as herbicides have also been investigated.
Elite transgenic event production and recovery relies heavily on the development of enabling technologies. Current methods in place for transformation of suspension cell aggregates is Agrobacterium- and whisker-mediated methods. Agrobacterium method shows a backbone integration rate of up to 67-90% making it a very inefficient process, where WHISKERS™ mediated transformation will not serve as a high throughput process (HTP). The PEG mediated method is used always demonstrated with protoplast and the protoplast of tobacco though easy to transform, it is not amenable easily for HTP transformation process due to the problems of cell wall regeneration.
The art appears to be silent regarding protocols for single-cell-suspension-culture-based transformation. There are several reports on protoplast-based protocols, but these are devoid of cell walls unlike single cell suspensions of plant cells as discussed below.